Browsing by Subject "prenylation"
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Item APPLICATIONS OF CHEMICAL PROTEOMICS IN DEFINING PRENYLATED PROTEOMES AND DISCOVERING NOVEL PRENYLATED PROTEINS(2021-04) Suazo, Kiall FrancisSince its development as a strategy to study biological systems, click chemistry has found its widespread use as a chemical tool in the discovery of post-translationally modified (PTM) proteins. In particular, the number of identified and validated lipid-modified proteins has dramatically increased over the last decade owing to the versatility and ease of use of this technique. Among these lipid PTMs, protein prenylation has benefited in uncovering new protein substrates, leading to the characterization and understanding of the functional role of this type of modification. This dissertation describes the development of a mass spectrometry-based chemical proteomics strategy that heavily relies on click chemistry to identify prenylated proteins in various systems, which allows for defining the prenylated proteomes and discovering novel prenylated proteins. The first chapter details a comprehensive literature review on the impact of click chemistry in revolutionizing the field of protein lipidation. Chapter 2 describes the development of chemical proteomics using probe analogue of isoprenoids to define the set of prenylated proteins in the malaria parasite, Plasmodium falciparum. Upon moving to eukaryotic systems, optimization of the probe and statin (enhancer of probe incorporation) treatments is described in Chapter 3. Our chemical proteomics approach was then successfully employed in various mammalian cell lines, enabling the validation of known and discovery of novel prenylated proteins. Moreover, the similarities and differences in brain-related cell lines were determined. Finally, an unusually prenylated protein, ALDH9A1, was discovered from these proteomic studies and tools in chemical and molecular biology were used to characterize and understand its occurrence. This dissertation highlights the advantages of using chemical proteomics in studying protein prenylation and contributes to the growing picture of protein lipid modifications.Item Solid-Phase Synthesis Of Prenylated Peptides Containing C-Terminal Esters: A Chemical Biology Tool For The Study Of Protein Prenylation(2015-09) Diaz-Rodriguez, VeronicaProtein prenylation is a post-translational modification that comprises the attachment of either a farnesyl or a geranylgeranyl isoprenoid. This covalent, irreversible modification has been studied extensively due to its importance for the proper cellular activity of numerous proteins. Due to appearance of mutated forms of farnesylated Ras in around 30% of all human cancers, substantial efforts have been focused on the development of FTase inhibitors (FTI). Despite numerous studies on the enzymology of the proteins involved in this pathway many questions remains about the protein prenylation process in cells. The mating pheromone a-factor is a farnesylated dodecapeptide found in the budding yeast S. cerevisiae which biosynthesis encompasses the same processing pathway than Ras proteins (farnesylation of C-terminal cysteine, C-terminal proteolysis and C-terminal methyl esterification). For mating, a-factor travels to the cell surface of the opposite mating cell were it binds and activate a membrane receptor. In this dissertation, our efforts in using a-factor as a model system to understand the role of prenyl groups in protein-protein interactions is described. First, we developed a method for the solid-phase synthesis of peptides containing the C-terminal cysteine esters found in mature prenylated proteins. Next, we have shown that not only methyl esters but also peptides containing ethyl, isopropyl and benzyl cysteine esters may be obtained with our strategy. Additionally, the approach was used to prepare a-factor and a-factor analogs that were tested for their biological activities. Our results indicate that this simple method can be used for the synthesis of a variety of C-terminal ester modified peptides that should be useful in studies of protein prenylation and other structurally related biological processes.